Lov-D acyltransferase mediated acylation

ABSTRACT

Methods for the improved acylation of chemical substrates using LovD acyltransferases, thioesters having acyl groups, and (i) thiol scavengers and/or (ii) precipitating agents are presented. An improved method for the production of simvastatin using (i) activated charcoal as a thiol scavenger and/or (ii) ammonium hydroxide as a precipitating agent is also presented.

1. CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a national stage application filed under 35USC §371 and claims priority of the international applicationPCT/US2010/0505253, filed Sep. 24, 2010, and U.S. provisional patentapplication 61/247,724, and 61/247,254 both filed Sep. 30, 2009, whichare hereby incorporated by reference herein.

2. REFERENCE TO SEQUENCE LISTING

The Sequence Listing concurrently submitted herewith under 37 CFR §1.821in a computer readable form (CRF) via EFS-Web as file name CX2-032.txtis incorporated herein by reference. The electronic copy of the SequenceListing was created on Sep. 24, 2010, with a file size of 52 Kbytes.

3. BACKGROUND

The invention relates to a process for forming simvastatin orsimvastatin precursors using improved LovD acyltransferase mediatedacylations.

Enzymes are biomolecules which catalyze the conversion of a chemicalsubstrate into a product and have been used in the chemical synthesis ofvaluable natural products and pharmaceuticals. Advantageously, enzymescan function to increase the rate of chemical conversion of a substrateto a product (by lowering the activation energy for the reaction) and todirect the placement of functional groups, i.e. regioselectively and/orstereoselectively placing functional groups onto a substrate. Enzymeactivity can be affected by other molecules known as inhibitors. Theseinhibitors function to decrease enzyme activity and can severely limitthe conversion or rate of conversion of a starting substrate intoproduct.

One particular group of useful enzymes includes the transferases.Transferases are enzymes that catalyze the transfer of a functionalgroup, for example, alkyl, acyl or phosphate groups, from a substrate(designated the donor and sometimes known as the coenzyme) to anothersubstrate (designated the acceptor). The enzyme thereby catalyzes areaction between chemical compounds that results in the loss offunctionality from the donor and a gain in functionality on theacceptor. The subclass of acyltransferases has been used, among otherthings, to regioselectively acylate chemical substrates such asmonacolin J to form simvastatin.

Simvastatin is a semisynthetic derivative of the natural productlovastatin, which can be isolated from the fermentation broth ofAspergillus terreus. Both lovastatin and simvastatin are cholesterollowering drugs that substantially lower the risk of heart disease amongadults. The gene cluster for lovastatin biosynthesis in A. terreus hasbeen described previously, for example, in U.S. Pat. No. 6,391,583.Encoded in the gene cluster is a 46 kD protein known as LovD, whichfunctions as an acyltransferase.

Once lovastatin is produced via fermentation in an A. terreus host,simvastatin can be produced from lovastatin via a semisynthetic route.After isolation and purification of lovastatin from the fermentationbroth, a typical semisynthesis can proceed by hydrolysis of the2-methylbutyrate side arm in the presence of base to yield theintermediate monacolin J. Monacolin J is the immediate precursor tosimvastatin. Following hydrolysis, the free acid is lactonized, the freehydroxyl at C13 is protected, and the C8 alcohol is acylated to providea protected analogue of simvastatin. Subsequent deprotection affordssimvastatin. See, e.g., WO 2007/139871.

Enzymatic transformations using lipases and esterases have also beeninvestigated as alternatives to chemical derivation. See, e.g., PCT WO2005/040107, PCT WO 94/26920 and T. G. Schimmel, et. al. in Appl.Environ. Microbiol. (1997) 63:1307-1311. Enzymatic variants suffer fromdecreased throughput of substrate, high loading requirements, slowenzyme conversion rate or poor enzyme turnover. Therefore, an enzymaticmethod of producing simvastatin, such as by selective acylation of theC8 hydroxy of monacolin J, which provides good to high yield withminimum isolation steps, good enzyme turnover and conversion rate,and/or reasonable loading requirements is important towards theefficient synthesis of simvastatin and additional statin analogs.

Accordingly, there has been a long-felt need for an enzymatic processwhich overcomes one or more limitations of the prior art, therebyproviding a method for the efficient and expedient acylation of chemicalsubstrates such as lovastatin or monacolin J using acyltransferases.

4. SUMMARY

It has been surprisingly discovered that the LovD acyltransferasemediated acylation of simvastatin precursors is an equilibrium processand that this equilibrium process may be adjusted in one or more ways toresult in higher conversion and/or rate of conversion of substrate toproduct.

It has also been surprisingly discovered that simvastatin may beprecipitated as an insoluble salt in the acylation of a simvastatinprecursor during a LovD acyltransferase mediated acylation reaction, andthat this results in a shift of equilibrium, thereby providing higherconversion and/or rate of conversion of substrate to product.

It has been surprisingly discovered that LovD acyltransferase may beinhibited by thiol byproducts produced during the acylation of monacolinJ hydroxy acid salt. More particularly, it has been discovered thatwhereas LovD acyltransferase can mediate the acylation of monacolin Jhydroxy acid salt into the corresponding simvastatin acid in high yield,the reaction rate of conversion may be improved by preventing theinhibition of the LovD enzyme by thiol byproduct. Accordingly, thepresent disclosure is, in one or more embodiments, directed to a methodof preventing the inhibition of the LovD acyltransferase enzyme byaddition of thiol scavengers. See FIG. 1.

Advantageously, it has been found that addition of thiol scavengers suchas activated charcoal improves the conversion rate of monacolin Jsubstrate to its acylated analogue when thioester compounds comprisingacyl moieties are used as co-substrates. The methods described hereinovercome one or more limitations of the prior art and satisfy a longfelt need for an efficient and expedient synthesis of simvastatin andsimvastatin precursors using LovD acyltransferase enzymes.

The present disclosure, in one or more embodiments, also provides amethod of producing simvastatin which overcomes the newly discoveredinhibition of LovD acyltransferase by thiol byproducts and which shiftsthe equilibrium toward reaction product. It has been further discoveredthat addition of a thiol scavenger such as activated charcoal preventsor reduces enzyme inhibition. This results in an improved enzymaticconversion of substrate to a target compound and thereby provides anadvantageous method for the production of simvastatin. In particularembodiments, methods and materials designed to take advantage of theimproved enzymatic conversion process are described. More particularly,the present disclosure, in one or more embodiments, is directed to amethod for the production of an enzymatically acylated chemicalsubstrate (simvastatin or a simvastatin precursor), wherein a thiolscavenger is utilized to prevent inhibition of the acyltransferaseenzyme. See FIG. 1 (illustrating an exemplary and generalized LovDacyltransferase reaction wherein thiol byproduct is sequestered).

In one or more embodiments, the method comprises the steps of combininga LovD acyltransferase enzyme in a reaction medium with (i) a thiolscavenger and/or (ii) a precipitating agent, a substrate comprising afree hydroxyl moiety, and a thioester comprising an acyl moiety. Theacyltransferase mediates the donation of an acyl moiety from thethioester to the free hydroxyl moiety, thereby producing the targetcompound. While not intending to be bound by any theory of operation, inembodiments wherein a thiol scavenger is utilized, the thiol scavengeris believed to sequester (bind or otherwise inactivate) the resultingthiol byproduct. The thiol byproduct may act as an enzyme inhibitor ofLovD, thereby reducing enzyme activity and conversion of the substrateto product. Enzyme inhibition may be a result of competitive binding ofthe inhibitor to the enzyme. Also, again while not intending to be boundby any theory of operation, in embodiments wherein a precipitating agentis utilized, it is believed that removal of product from the reactionmedium by precipitation results in a favorable shifting of the reactionequilibrium. Removal of thiol byproduct may also provide a favorableshifting of the equilibrium.

Embodiments described herein also include methods for generatingsimvastatin with a minimum of chemical steps using an improved LovDacyltransferase reaction. Advantageously, the improved acyltransferasereaction can optionally be combined with additional chemical steps, suchas in “one-pot” chemical synthesis methods, to provide a method ofproducing simvastatin from lovastatin without isolation and purificationof the monacolin J intermediate. For example, lovastatin may behydrolyzed to monacolin J and the crude reaction product used as thesubstrate for the enzyme mediated acylation reaction. The presentdisclosure also provides methods and materials designed to takeadvantage of the improved LovD enzymatic process, such as that by whichlovastatin is made, in order to produce related compounds such as thepravastatin derivative huvastatin.

Those of skill in the art will understand that the disclosure providedherein allows artisans to produce a wide variety of embodiments. In oneexemplary embodiment, simvastatin is produced by combining togethermonacolin J or a monacolin J derivative (preferably monacolin J hydroxyacid), a thioester that donates an acyl moiety to the C8 hydroxyl groupof monacolin J (or derivative) in the presence of a LovDacyltransferase, (i) a thiol scavenger (comprising, for example,activated charcoal) and/or (ii) a precipitating agent (such as ammoniumhydroxide (NH₄OH)), and a LovD acyltransferase. The LovD acyltransferasefunctions to mediate the transfer of an acyl group from the thioester toregioselectively acylate the C8 hydroxyl group of monacolin J (orderivative), thereby producing simvastatin. The resulting byproductthiol is sequestered (bound or deactivated by the thiol scavenger) whenthiol scavenger is present. Reaction product is precipitated when aprecipitating agent is present. Advantageously, reaction rate isnoticeably or markedly improved.

A related embodiment provides a method of making simvastatin, comprisingthe steps of combining together lovastatin, a thioester that donates anacyl moiety to the C8 hydroxyl group of monacolin J in the presence of aLovD acyltransferase, (i) a thiol scavenger (comprising, for example,activated charcoal) and/or (ii) a precipitating agent (such as ammoniumhydroxide (NH₄OH)) and a LovD acyltransferase. In this embodiment of themethod, the LovD acyltransferase is allowed to hydrolyze lovastatin intomonacolin J prior to the transfer of an acyl group from the thioestervia a regioselective acylation of the C8 hydroxyl group of monacolin Jusing the acyltransferase, thereby providing simvastatin. The resultingbyproduct thiol is sequestered (bound or deactivated by the thiolscavenger) when thiol scavenger is present. Reaction product isprecipitated when a precipitating agent is present. Advantageously,reaction rate is improved and high conversions achieved.

The methods and materials described herein that are used to makesimvastatin can be adapted to produce other compounds including thosethat are structurally similar to simvastatin, for example huvastatin. Inthis context, a method of making huvastatin is provided that comprisesthe steps of combining together hydrolyzed pravastatin tetra-ol, athioester that donates an acyl moiety to the C8 hydroxyl group ofhydrolyzed pravastatin tetra-ol in the presence of a LovDacyltransferase, (i) a thiol scavenger (comprising, for example,activated charcoal) and/or (ii) a precipitating agent (such as NH₄OH),and a LovD acyltransferase. The LovD acyltransferase is allowed to usean acyl group from the thioester to regioselectively acylate the C8hydroxyl group of hydrolyzed pravastatin tetraol, so that huvastatin ismade. The thiol scavenger (when present) is believed to again act bysequestering the thiol byproduct, thereby preventing inhibition of theLovD enzyme, resulting in an improved enzyme reaction rate and ashifting of the equilibrium towards reaction product. The precipitatingagent (when present) is believed to shift the equilibrium towardreaction product by removing reaction product from solution. In yetanother embodiment, huvastatin can be made directly from pravastatin.Pravastatin is combined with the LovD acyltransferase to hydrolyzepravastatin to the intermediate hydrolyzed pravastatin tetra-ol.Acylation by the LovD acyltransferase then can proceed upon addition of(i) a thiol scavenger and/or (ii) a precipitating agent, and a thioestercomprising an acyl moiety.

In typical embodiments of the various methods described herein, thefunctional group donor is an acyl donor. Thioesters are one highlypreferred group of donor compounds that can donate an acyl moiety to achemical substrate in the presence of a LovD acyltransferase and ascavenging compound(s). For example, the C8 hydroxyl group of monacolinJ may be acylated by a thioester in the presence of a LovDacyltransferase. A variety of such thioesters are disclosed herein. See,e.g., FIG. 4 (illustrating some preferred thioester compounds). Inaddition to the preferred thioesters of FIG. 4, other preferredthioesters include butyryl-thioesters, N-acetylcysteamine thioesters ormethylthioglycolate thioesters. Biologically derived thioester compoundssuch as acetyl coenzyme A (Acetyl CoA) may be used in one or moreembodiments. Acetyl CoA comprises a thioester between coenzyme A (athiol) and acetic acid (an acyl group carrier). Accordingly, othercompounds comprising acyl donors include acyl-CoA, butyrlyl-CoA,benzoyl-CoA, acetoacetyl CoA, D-hydroxybutyryl-CoA, malonyl-CoA andpalmitoyal-CoA. The thioester group may comprise one or more functionalgroups such as, for example, an alkyl linker chain, an alkyl group(s),aryl group(s), esters, amides, sulfonates, phosphates, and so on.Preferably, the thioester comprises a short alkyl chain terminated in anester or amide, or the thioester may comprise only an alkyl chain. Otherfunctional group donors with the capacity to donate an acyl group arealso contemplated as suitable donors. Other acyl donating groups whichform thiol byproducts upon LovD-mediated acylation of the targetsubstrate would benefit from the methods of one or more embodiments ofthe present disclosure, in particular, the addition of scavengers whichsequester one or more thiol moieties, and/or the addition ofprecipitating agents.

Optionally, the thioester comprises one or more short (C₁-C₂), medium(C₃-C₆), or long (>C₇) chain length acyl group moieties which may bebranched, unbranched, or cyclic. It is contemplated that in someinstances the acyl group moieties may be functionalized. Somerepresentative thioesters areα-dimethylbutyryl-5-methyl-mercaptopropionate (also known as methyl3-(2,2-dimethylbutanoylthio)propanoate, DMB-S-MMP),dimethylbutyryl-5-ethyl mercaptopropionate (DMB-S-EMP),dimethylbutyryl-5-methyl thioglycolate (DMB-S-MTG) anddimethylbutyryl-5-methyl mercaptobutyrate (DMB-S-MMB). In anillustrative and preferred embodiment, the thioester isS-2-acetamidoethyl 2,2-dimethylbutanethioate, S-acetamidomethyl2,2-dimethylbutanethioate, methyl 2-(2,2-dimethylbutanoylthio)acetateand/or methyl 3-(2,2-dimethylbutanoylthio)propanoate. Thioesters may bemade by methods known in the art. In an exemplary process, the highlypreferred thioester DMB-S-MMP may be prepared by acylation of methyl3-mercaptopropanoate with 2,2-dimethylbutanoyl chloride in the presenceof N,N-diisopropylethylamine (DIPEA). Other acyl thioesters may be madeby utilizing other acyl chlorides (or halides) and inorganic or organicbases in the acylation of methyl 3-mercaptopropanoate.

Acyltransferase enzymes useful in the methods described herein are LovDacyltransferases. The LovD acyltransferase may be a wild-type LovDenzyme obtainable from A. terreus, or a mutant thereof, such as forexample, the mutants described in Biotechnol Bioeng, 2009 Jan 1;102(1):20-8. Specific exemplary LovD acyltransferases that catalyze themethods described herein with greatly increased reaction rates and yieldas compared to wild-type LovD acyltransferase from A. terreus that canbe advantageously used in the methods described herein are described inApplication No. 61/247,253, titled “LovD Variants and Their Uses,” filedSep. 30, 2009 and application Ser. No. 12/890,134 filed Sep. 24, 2010.Suitable LovD acyltransferases are also described below in Section 6(See, for example, Table 2).

The methods disclosed herein utilize one or more scavenger compounds oragents. Scavenger compounds adapted to scavenge thiol compounds include,for example, activated charcoal, isatoic anhydride, fluorous2,4-dichloro-1,3,5-triazines (F-DCTs), vinyl ethers, dihydropyran,N-ethylmaleimide, p-(chloromercuri)benzoate, copper ions, and variantsthereof. Thiol scavengers may be adapted by incorporation onto asolid-support and may be used alone or in combination with otherscavengers including non-thiol scavengers, i.e. scavengers which act tobind or deactivate other functional groups which inhibit enzymeactivity. In a preferred embodiment, activated charcoal is utilized.

The methods disclosed herein may also utilize in addition to or inexclusion to one or more scavenger compounds or agents, one or moreprecipitating agents. Preferred precipitating agents are compounds whichfunction to donate a counterion to the acylated substrate which renderthe compound insoluble in the reaction medium. For example, ammoniumhydroxide functions to donate an ammonium counterion (NH₄ ⁺).

In certain embodiments described herein, the methods result in improvedLovD enzyme activity. Improved enzyme activity corresponds to improvedenzyme stability, improved enzyme rate, i.e. the rate at which substrateis modified, e.g., acylated, and/or improved enzyme loadingrequirements, e.g., the amount of enzyme required to achieve a givenconversion in a given timeframe. In an exemplary and preferredembodiment, the enzyme rate is improved. For example, the rate at whichacylation of monacolin J hydroxy acid, sodium salt to the correspondingsimvastatin hydroxy acid, sodium salt is effected can be improved, i.e.complete equilibrium conversion, meaning the time at which no moresubstrate is acylated, occurs in less time as exemplified in theexamples given below.

Certain embodiments of the methods for making simvastatin and relatedcompounds using the improved LovD acyltransferase reaction may includefurther steps to purify these compounds. For example, embodiments of thedisclosure can include at least one purification step comprising lysisof cells of an isolated organism present in the combination. Embodimentscan also include at least one purification step comprisingcentrifugation of cells or cell lysates of an isolated organism presentin the combination. Moreover, embodiments can include at least onepurification step comprising precipitation of one or more compoundspresent in the combination. One embodiment of a precipitation stepcomprises the precipitation of a free acid form of simvastatin.Optionally in such embodiments, one can then convert this free acid formof simvastatin to a simvastatin salt. Embodiments of the disclosure canalso include at least one purification step comprising filtration orchromatography of one or more compounds present in the combination. Inaddition, embodiments can include at least one analysis step comprisingspectrometry such as proton or carbon NMR or chromatography such asflash chromatography, thin-layer chromatography, gas chromatography,and/or high performance liquid chromatography (HPLC).

Additional embodiments are discussed below.

5. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a generalized scheme of the LovD acyltransferasemediated acylation of a hydroxyl group by a donor compound andsubsequent “scavenging” of the thiol byproduct;

FIG. 2 illustrates the hydrolysis of lovastatin (100), which can behydrolyzed chemically or enzymatically to produce (a) LovD hydrolyzedlovastatin (110), (b) monacolin J hydroxy acid (120) or (c) monacolin Jhydroxy acid, sodium salt (140);

FIG. 3 illustrates various acyltransferase mediated acylations ofmonacolin J hydroxy acid and its salts;

FIG. 4 illustrates some representative thioesters comprising an acylgroup;

FIG. 5 illustrates lovastatin (100) and simvastatin (160) with chemicalnumbering and labels; and

FIG. 6 illustrates the synthesis of simvastatin (160) from lovastatin(100) via a LovD hydrolyzed lovastatin (110).

6. DETAILED DESCRIPTION

The LovD acyltransferase mediated acylation of statin precursors such asmonacolin J or analogues and derivatives thereof is an equilibriumprocess. This means that reaction products may inhibit the additionalformation of product. The equilibrium process can be shifted towardreaction product by either (i) increasing thioester loading; (ii)removing/sequestering thiol byproduct; or (iii) product precipitation.The processes of the present disclosure utilize one or more equilibriumshifting techniques to improve the percent conversion of substrate toproduct and/or the reaction rate. For example, in some embodiments,increased thioester loading is utilized to improve reaction yield/rate.In other embodiments, thiol byproduct is scavenged to improve reactionyield/rate. In yet other embodiments, product is precipitated duringreaction, thereby improving reaction yield/rate. Equilibrium shiftingtechniques may be used in combination. For example, in some embodiments,additional thioester co-substrate and product precipitation are utilized(with or without the addition of a thiol scavenger).

The present disclosure, in one or more embodiments, provides a novelprocess for the improved acylation of chemical substrates using LovDacyltransferase. Preferred embodiments include a method of improving theacyltransferase activity of LovD or its variants by addition ofscavenger compound(s). Another preferred embodiment comprises the use ofa precipitating agent. Yet additional preferred embodiments include amethod of producing an acylated chemical substrate using LovDacyltransferase, a thioester comprising an acyl moiety and (i) ascavenging compound(s) and/or (ii) a precipitating agent. Exemplary ofone or more preferred embodiments is an improved process for theacylation and hence production of simvastatin from either lovastatin ormonacolin J (or its hydroxy acid salts) using the LovD acyltransferase,a thioester comprising an acyl moiety, and (i) a scavenging compound(s)and/or (ii) a precipitating agent.

One exemplary and preferred scavenger compound is activated charcoal.Without being tied to any one theory of operation, it is hypothesizedthat activated charcoal traps LovD enzyme inhibiting thiol byproductsand/or converts (“deactivates”) thiol byproducts into disulfides that donot interfere with enzyme activity. Other thiol scavengers which do notinterfere with enzyme activity and which bind or deactivate thiolbyproducts are also preferred.

6.1. Definitions

As used herein, the following terms are intended to have the followingmeanings:

“Lovastatin” (Mevacor®) is a fungal polyketide produced by Aspergillusterreus. See, e.g., A. W. Alberts, J. et. al., Proc. Natl. Acad. Sci.U.S.A., 1980, 77, 3957-3961 and A. Endo, J. Antibiot. 1980, 33, 334-336;and J. K. Chan, et. al., J. Am. Chem. Soc. 1983, 105, 3334-3336; Y.Yoshizawa, et. al., J. Am. Chem. Soc. 1994, 116, 2693-2694. It is apharmaceutically important compound because of its potent inhibitoryactivities towards hydroxymethylglutaryl coenzyme A reductase (HMGR),the rate-limiting step of cholesterol biosynthesis, and therefore it iswidely used in the treatment of hyperlipidemia, hypercholesterolemia,and the like. See FIG. 5 for chemical numbering and labeling oflovastatin (100).

“Simvastatin” is an analog of lovastatin. It is favored over lovastatinbecause of the absence of adverse side effects and its highabsorbability in the stomach. Also, it has been reported thatsimvastatin prevents and reduces the risk of Alzheimer's disease (AD) byretarding the production of Ab42, a β-amyloid protein associated withAD. It is known in the art that simvastatin can be syntheticallyprepared. See, e.g., U.S. Pat. Nos. 4,444,784, 4,582,915, 5,393,893,5,763,646 and 5,763,653, EP Pat. No. 299,656 and Intl. Pat. Pub. No. WO99/45003. See FIG. 5 for chemical numbering and labeling of simvastatin(160).

“Lovastatin derivatives” as used herein comprises lovastatin derivativesor precursors, for example: pravastatin, huvastatin, and simvastatin.

“Monacolin J variants” refers to monacolin J variants disclosed in theart, for example, the hydrolyzed pravastatin tetra-ol or6-hydroxy-6-desmethylmonacolin J and the like. In certain embodiments ofthe disclosure, “monacolin J variants” refers to monacolin J compoundshaving substitutions at the C6 position or to the hydroxy acid form ofmonacolin J (and salts thereof).

Skilled artisans will appreciate that lovastatin, monacolin J andsimvastatin, as well as their analogues and derivatives can exist invarious forms including acid, ester, amide and lactone forms. The acid,ester, amide and lactone forms can also be in the form of salts. Theacid (R=—OH), ester (R=—O(alkyl)), amide (R=—N(alkyl)₂) and lactoneforms of these compounds are illustrated below. Unless stated otherwise,“lovastatin” as used herein includes the acid, ester, amide, lactone andsalt forms, “monacolin J” as used herein includes the acid, ester,amide, lactone and salt forms and “simvastatin” as used herein includesthe acid, ester, amide, lactone and salt forms. These forms can be usedin the methods described herein.

The salts of these compounds (e.g., pharmaceutically acceptable saltsknown in the art) can occur both as a free acid as well as a sodium,potassium, ammonium, or other salts derived from metals of group I orgroup II including alkaline earth elements or other metallic salts.Organic salts may also be utilized including, for example, ammonium andtriethanolamine salts. Such salts may be found in combination; forexample, a simvastatin salt could be a combination of sodium andpotassium salts with simvastatin.

“Aspergillus terreus” or “A. terreus” is a filamentous ascomycetecommonly found in soil. A variety of A. terreus strains are known in theart, for example, those deposited as ATCC 20542 and ATCC 20541.

“LovD acyltransferase” as used herein refers to those polypeptides thatcan use a thioester to regiospecifically acylate the C8 hydroxyl groupof monacolin J or 6-hydroxy-6-des-methyl monacolin J so as to producesimvastatin or huvastatin, respectively. See, e.g., Xie et al., 2006,“Biosynthesis of Lovastatin Analogs with a Broadly SpecificAcyltransferase,” Chem. Biol. 13:1161-1169. LovD acyltransferasesinclude, by way of example and not limitation, the wild-type LovDacyltransferase obtainable from A. terreus (amino acid sequence providedherein as SEQ ID NO:2), as well as mutants, variants, and truncatedforms thereof, as will described in more detail below).

As disclosed herein, an “acyl donor” or “acyl carrier” is a compoundhaving an acyl group that can be transferred to a target substrate, suchas, for example, simvastatin and/or a simvastatin precursor or a relatedcompound. Typically, an “acyl donor” or “acyl carrier” is a thioesterthat donates (as mediated by an acyltransferase) an acyl moiety to aspecific region on a target molecule, such as, for example, the C8hydroxyl group of monacolin J. A wide variety of such agents are knownin the art to have this activity. See, e.g., WO 2007/139871. In additionto those known in the art and further shown by the instant disclosure tohave this activity, any potential acyl donor/carrier known in the art(or synthesized de novo) having an ability to acylate a target substratevia an acyltransferase, such as C8 of monacolin J (thereby producingsimvastatin), can be easily identified by comparative experiments withthe acyl donors disclosed.

Other examples of acyl donors include, but are not limited to,α-dimethylbutyryl-SNAC, acyl-thioesters, acyl-CoA, butyryl-CoA,benzoyl-CoA, acetoacetyl-CoA, β-hydroxybutyryl-CoA, malonyl-CoA,palmitoyal-CoA, butyryl-thioesters, N-acetylcysteamine thioesters(SNAC), methyl-thioglycolate (SMTG), benzoyl-SNAC, benzoyl-SMTG orα-S-methylbutyryl-SNAC. These compounds can be produced naturally orsynthetically, and, in some cases, can penetrate a cell membrane. Anumber of these compounds can be added to a reaction medium comprisingLovD and monacolin J to produce simvastatin, for example.

“Acyl-SNAC” as used herein refers to α-dimethylbutyryl-SNAC. As is knownin the art, acyl-SNAC can penetrate a cell membrane under in vivoconditions. LovD can use acyl-SNAC as a substrate to initiate thereaction from monacolin J to simvastatin by regiospecifically acylatingthe C8 hydroxyl group of monacolin J. Acyl-SNAC can donate its acylgroup to LovD.

“Alkyl” means an unsubstituted or substituted alkyl group. An alkyl canbe, for example, cyclic, acyclic, branched, or linear.

“Aryl” means an unsubstituted or substituted aryl group and may compriseheterocycles.

“A thioester comprising an acyl moiety” means a thioester having an acylmoiety which undergoes a mediated transfer to a substrate when treatedunder appropriate conditions with an acyltransferase. For example, acompound of general formula R—C(═O)—SR′ wherein R is an alkyl or aryland R′ is variable comprises an acyl group as R—C(═O)—.

“A suitable reaction medium” is a solvent system, mono or biphasic,which allows the components of the reaction mixture to combine andproduce product. Exemplary reaction mediums include ether, ethylacetate, hexanes, tetrahydrofuran, methanol, isopropanol, acetone,dimethylformamide, dichloromethane, chloroform, tert-butyl methyl ether(MTBE), water, acetonitrile, and combinations thereof. Reaction mediumsmay be buffered to maintain a specific pH or pH range such as byaddition of acids, bases, or salts. For example, one exemplary reactionmedium is a triethanolamine buffered aqueous suspension of reactants.Another exemplary reaction medium is ethyl acetate with the addition ofmethanol. Preferably, reaction mediums are maintained at controlledtemperatures, under inert atmospheres such as argon or nitrogen, andwith mechanical or magnetic stirring.

A “scavenger compound” is a compound which binds, chemical modifies toan inert form (relative to enzyme activity) or removes a byproductformed during an acyltransferase mediated acylation reaction. Theprocess of binding or chemically modifying a compound to an inert formis also known as sequestering.

“Activated charcoal,” also called activated carbon or activated coal, isa form of carbon that has been processed to provide a very large surfacearea. A suitable form of activated charcoal is represented by Fluka®puriss.p.a. powdered activated charcoal (CAS No. 7440-44-0,iodine-adsorption (0.05 mol I₂/I) of >70 mL/g).

LovD “Acyltransferase mediated acylation” means acylation of a substrateis mediated by the LovD acyltransferase. Without being bound by any onetheory of operation, it is believed that LovD acyltransferase isacylated by a thioester compound, accepting the acyl-moiety of thethioester and producing a thiol byproduct. Subsequently, a free hydroxylmay then accept the acyl-moiety attached to the LovD acyltransferase,thereby becoming acylated itself and regenerating the active LovDacyltransferase. It has been discovered that the production of thiolcompounds during the acyltransferase reaction results in decreasedactivity of the enzyme. In particular, it has been found that thiolcompounds inhibit the rate of enzyme conversion of substrate to target.

“Precipitating agent” is a chemical agent which induces theprecipitation of a compound or which converts a starting material into acompound which becomes insoluble after subsequent reaction.Precipitation is understood to mean the deposition of insoluble matterin a reaction medium, i.e. the formation of a solid in a solution duringa chemical reaction. A precipitation agent functions by converting anormally soluble compound into a compound which undergoes precipitationin the reaction medium or which undergoes precipitation in the reactionmedium upon further conversion to a target. A precipitating agent mayalso function to maintain or adjust solution pH. For example, ammoniumhydroxide may be utilized as a precipitating agent in that monacolin Jhydroxy acid may be converted to its ammonium salt prior to acylation.Acylation in a suitable reaction medium then results in an insolubleproduct. Ammonium hydroxide also functions to adjust the pH of thesolution.

“Insoluble,” “Substantially Insoluble,” and “Partially Insoluble” referto the ability of a solute to form a solution in a liquid solvent.Solubility is, unless otherwise stated, measured at room temperature andpressure. A substance is insoluble if for a given solvent system, i.e. amedium comprising one or more solvents, complete dissolution underreaction concentrations is not achievable. A substance is substantiallyinsoluble if no more than 5% of the substance undergoes dissolutionunder reaction concentrations in a given solvent system. A substance ispartially insoluble if more than 5% but less than 95% of the substanceundergoes dissolution under reaction concentrations in a given system. Aprecipitating agent achieves at least the partial insolubility of acompound.

6.2. Detailed Description

The present disclosure, in one or more embodiments, provides methods andmaterials designed to take advantage of an improved LovD acyltransferasereaction. More particularly, one or more embodiments provide a methodfor the production of a LovD acylated chemical substrate, wherein athiol scavenger is utilized to prevent inhibition of the LovDacyltransferase enzyme. See FIG. 1. In a typical embodiment, the methodcomprises the steps of combining LovD acyltransferase enzyme in areaction medium comprising a thiol scavenger, a substrate comprising afree hydroxyl moiety and a thioester. LovD mediates the donation of anacyl moiety from the thioester to the free hydroxyl moiety, therebyproducing the target compound. The thiol scavenger sequesters orotherwise inactivates the resulting thiol byproduct, which is believedto act as an enzyme inhibitor, thereby allowing improved enzymaticconversion of the substrate to product.

In other embodiments, a method for the production of a LovD acylatedchemical substrate, wherein a precipitating agent is utilized whichrenders the final product insoluble in the reaction medium, isdisclosed. In a typical embodiment, the method comprises the steps ofcombining LovD acyltransferase enzyme in a reaction medium comprising aprecipitating agent, a substrate comprising a free hydroxyl moiety and athioester. LovD mediates the donation of an acyl moiety from thethioester to the free hydroxyl moiety, thereby producing the targetcompound. The target compound is rendered insoluble as a result of theprecipitating agent. Removal of the target compound by precipitationresults in a favorable shift in the equilibrium of the reaction.

The present disclosure also provides, in one or more embodiments, amethod of acylating a chemical substrate comprising a free hydroxylcompound using LovD acyltransferase, (i) a thiol scavenger and/or (ii) aprecipitating agent, and a compound of general formula (I):

wherein:

-   -   R₁ represents an aryl or alkyl group; and    -   Y is (a) —(CH₂)_(n)—NR₂—(CO)—R₃; or (b) —(CH₂)_(n)—(CO)O—R₃;        or (c) —(CH₂)_(n)—H or an optionally substituted alkyl or        optionally substituted aryl group;    -   wherein n is an integer from 1-10 and R₂ and R₃ are,        independently, an alkyl or aryl group.

The method comprises combining the free hydroxyl containing compoundinto a suitable reaction medium with the LovD acyltransferase, (i) athiol scavenger and/or (ii) a precipitating agent, and a thioestercomprising an acyl moiety. For example, the free hydroxy containingcompound can be added into a round bottom flask comprising a suitablereaction medium, such as an aqueous solution buffered to within pH 6-11and often pH 7-10 by addition of a base, e.g., triethanolamine, ammoniumhydroxide, sodium hydroxide, or other inorganic or organic bases. Understirring, the reaction vessel can be charged with the LovDacyltransferase enzyme, (i) a thiol scavenger and/or (ii) aprecipitating agent and then the thioester. The reaction can bemonitored to follow conversion of the substrate, such as by HPLC,thin-layer chromatography, or other suitable methods. As appropriate,additional enzyme or other reactant (e.g., thioester) may be added toeffect optimal conversion. Workup, such as by appropriate quenching whennecessary, and extraction of the target compound or direct filtration ofthe product may be effected when the desired conversion has beenachieved. Filtration, such as through Celite® and drying of organicextracts, such as by addition of sodium sulfate or magnesium sulfate orby azeotropic drying, may also be performed. Extracts containing theproduct may be concentrated by appropriate methods such as under reducedpressure.

The present disclosure also provides, in one or more embodiments, amethod of acylating a chemical substrate comprising a free hydroxylcompound using LovD acyltransferase, (i) a thiol scavenger and/or (ii) aprecipitating agent and a compound of general formula (II):

wherein:

-   -   R₁ and R₂ represent, independently, an aryl or alkyl group; and    -   Y is —(CH₂)_(n)— or an optionally substituted alkyl or        optionally substituted aryl group;    -   wherein n is an integer from 1-10.

The method comprises combining the free hydroxyl containing compoundinto a suitable reaction medium with the LovD acyltransferase, (i) athiol scavenger and/or (ii) a precipitating agent and a thioestercomprising an acyl moiety. The chemical substrate can be lovastatin,monacolin J (or its hydroxy acids or hydroxy acid salts) or analoguesthereof comprising a free hydroxyl group. The present disclosure alsoprovides, in one or more embodiments, a method of acylating a chemicalsubstrate comprising a free hydroxyl compound using LovDacyltransferase, (i) a thiol scavenger and/or (ii) a precipitating agentand a compound of general formula (III):

wherein:

-   -   R₁ and R₂ represent, independently, an aryl or alkyl group; and    -   Y is —(CH₂)_(n)— or an optionally substituted alkyl or        optionally substituted aryl group;    -   wherein n is an integer from 1-10.

The method comprises combining the free hydroxyl containing compoundinto a suitable reaction medium with LovD acyltransferase, (i) a thiolscavenger and/or (ii) a precipitating agent and a thioester comprisingan acyl moiety. The chemical substrate can include lovastatin, monacolinJ (or its hydroxy acids or hydroxy acid salts) and analogues thereofcomprising a free hydroxyl group. The present disclosure also provides,in one or more embodiments, a method of acylating a chemical substratecomprising a free hydroxyl compound using LovD acyltransferase, (i) athiol scavenger and/or (ii) a precipitating agent and a compound offormula (IV):

The present disclosure comprises, in one or more embodiments, a methodof acylating lovastatin or monacolin J (or its hydroxy acids or hydroxyacid salts) and analogues thereof using LovD acyltransferase, (i) athiol scavenger comprising activated charcoal and/or (ii) aprecipitating agent comprising ammonium hydroxide, and a compound offormula (IV), comprising combination of the reactants in a suitablereaction medium, thereby allowing LovD to produce acylated forms oflovastatin or monacolin J.

As shown in table 1, thioesters as used in the present disclosure haverelative rates of reactivity, meaning some thioester compounds increasereaction rate. Any one or more compounds shown in Table 1 may be used asthioesters in the methods of the present disclosure. Compound IV is onepreferred thioester as its relative activity is high.

TABLE 1 Relative Com- activity at pound Structure 24 h (%) IV

++++ V

++ VI

+ VII

++ VIII

+ IX

+++ + = 0-1 ++ = 1-10 +++ = 10-75 ++++ = 75-100

The present disclosure also provides, in one or more embodiments, amethod of improving the enzymatic activity of LovD. The method comprisesthe addition of (i) a scavenger, preferably a thiol scavenger, and mostpreferably activated charcoal and/or (ii) a precipitating agent,preferably an agent which renders the product substantially insoluble,and more preferably ammonium hydroxide. In an exemplary embodiment,monacolin J hydroxy acid, sodium salt is acylated using a LovD or LovDvariant enzyme using a thioester in the presence of activated charcoal.In yet another exemplary embodiment, a reaction medium is charged with75 g/L of monacolin J hydroxy acid, sodium salt, 1.5 g/L of enzyme (LovDor equivalent amount of LovD variant), 1.7 equivalents (relative to thesubstrate) of thioester, and 10 g/L of activated charcoal. The reactionmay be run using a 200 mM triethanolamine buffer at pH 9.0 at roomtemperature. In the exemplary example, >98% of substrate is converted toacylated product within 24 hours. The relative concentrations of thereactants in the exemplary example are typical of one preferredembodiment.

In some embodiments, activated charcoal is added in sufficient amount tosequester at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, andat least 90% of thiol byproduct, preferably in sufficient amount tosequester 95% of thiol byproduct, and most preferably in sufficientamount to sequester at least 98% of thiol byproduct. Activated charcoalmay also be added in proportion to the amount of solvent; for example,2-20 grams of activated charcoal may be added per liter of solvent.Preferably, 5-15 grams of activated charcoal are added per liter ofsolvent. Most preferably, about 10 grams of activated charcoal are addedper liter of solvent. Addition of activated charcoal in one embodimentis made prior to the addition of both enzyme and donor molecule.Clarification of a substrate, such as during the workup of a reaction,may sometimes involve the use of activated charcoal. A person of skillin the art will appreciate that the use of activated charcoal in thismanner does not provide an improved enzymatic reaction because maximalconversion has already been achieved and the advantage of increasedenzymatic rate is no longer achievable.

Precipitating agents can produce compounds with reduced solubilityincluding salts. Various salts can be used including salts containingions of alkali metals and alkaline earth metals such as calcium, barium,lithium, and magnesium. An exemplary precipitating agent may include,for example, calcium hydroxide. Amine salts can also be used includingthose formed from primary, secondary, tertiary amines, aromatic amines,quaternary ammonium salts, and polyamines thereof. Sequestering agents,including ion exchange resins and other polymeric species, may also beused to effect removal of product from solution.

In another exemplary embodiment, 25 to 200 g/L, also 30 to 150 g/L, andoften 50 to 100 g/L (preferably about 75 g/L) of substrate are added toa reaction medium. In addition, 0.25 to 10 g/L, also 0.3 to 8 g/L andoften 0.5 to 6 g/L (preferably 0.75 g/L) of LovD enzyme or variant, and1.0 to 5, also 1.0 to 2.5 and often 1.05 to 1.7 (preferably 1.1)equivalents of thioester are added. The pH is controlled by, forexample, a pH stat, which maintains the pH of the system at about 8.5 to9.5 and preferably 9.0 by titration with ammonium hydroxide. Ammoniumhydroxide also functions as a precipitating agent. In preferredembodiments, no buffer is utilized. A typical solvent system may bewater. The reaction is preferably run with stirring at room temperature.

In an exemplary embodiment, simvastatin is produced from lovastatincomprising a first hydrolysis step. Hydrolysis may be effected toprovide monacolin J as the free hydroxy acid or the sodium salt of thehydroxy acid. See FIG. 2. Hydrolysis may also be effected in a mannerwhich provides monacolin J as an ammonium salt.

Monacolin J as the hydroxy acid may be (i) converted to one or moresalts such as its corresponding sodium or ammonium salts; and then (ii)acylated to provide a simvastatin precursor compound. When a sodium orother non-ammonium salt of the simvastatin precursor compound is formed,for example, compound 150 of FIG. 3, then the non-ammonium salt may beconverted to an ammonium salt (155). Lactonization of the ammonium salt(155) provides simvastatin (160). Alternatively, when an ammonium saltof monacolin J is used, monacolin J ammonium salt may be acylateddirectly to the ammonium salt simvastatin precursor (155). Some of thevarious routes to simvastatin are illustrated in FIG. 3.

Acyltransferase may also be used to effect one or more hydrolysis stepsin addition to the acylation of lovastatin. For example, LovDacyltransferase may be used to effect hydrolysis of lovastatin into aLovD hydrolyzed lovastatin (monacolin J lactone) and acylation of thefree hydroxyl at C8. See FIG. 6.

In practicing the methods of the disclosure, various amounts ofsubstrate, reactant, and reaction conditions may be used to carry outthe methods of one or more embodiments. Adjustable parameters includethe concentration of substrate in solution, the number of equivalents ofthe acyl donor relative to the substrate, buffer type and concentration,weight loading, atmospheric conditions, such as partial pressure and gastype, e.g., air, nitrogen, oxygen, argon, etc., the amount of thiolscavenger (if present), the amount of precipitating agent (if present),reaction pH, temperature, and stir-rate, the presence or absence ofco-solvent, reaction time, and the manner in which the substrate isisolated or prepared for further reaction.

In one or more embodiments, monacolin J hydroxy acid, sodium salt isreacted at a substrate loading between about 1 to 250 g/L, often 1 g/Lto 150 g/L or 50 g/L to 150 g/L. Preferably, monacolin J hydroxy acid,sodium salt is reacted at a substrate loading between 75 g/L and 150g/L. Most preferably, monacolin J hydroxy acid, sodium salt is reactedusing a substrate loading of about 75 g/L. A skilled artisan willappreciate that the substrate loading values may readily be convertedinto a molarity, wherein molarity is a measure of the concentration of asolute in solution. For example, a 75 g/L loading of the monacolin Jhydroxy acid (MJ), sodium salt has an approximate molarity of 0.21 M (75g/L MJ×(1 mol/360.42 g MJ)). In other embodiments, substrates other thanmonacolin J hydroxy acid, sodium salt may be acylated according to themethods of the disclosure. Preferably, such acylations will occur with asubstrate concentration having a molarity within the ranges given formonacolin J hydroxy acid, sodium salt. For example, a highly preferredreaction condition for acylation of a substrate is a substrate molarityof about 0.21 M.

The reaction can be carried out under a variety of different reactionconditions. Typically the reaction is carried out comprising about 0.2to 10 g/L, often 0.25 to 5 g/L, variant LovD polypeptide, from about 1to 250 g/L, often 50 to 150 g/L, monacolin J substrate (or a saltthereof) and from about 1 to 10 equiv, often 1 to 2 equiv,α-dimethylbutyryl thioester co-substrate. The reaction is typicallycarried out in an aqueous buffer (0 to 300 mM, preferably 50 to 300 mM,more preferably 200 mM) having a pH in the range of pH 7.5 to 10.5,often pH 8.0 to 9.5 or pH 8.5 to 9.5. The identity of the buffer is notcritical. Suitable buffers include, but are not limited to,triethanolamine (TEA), potassium phosphate, or a buffer may not be used.

Aqueous co-solvent systems can also be used. Such co-solvents willtypically include from about 1 to 10% of a polar organic co-solvent.Suitable polar organic co-solvents include, but are not limited to,MeCN, DMSO, isopropyl alcohol (IPA), dioxane, THF, acetone, and MeOH.

In one or more embodiments, the acyl donor is charged to the reactionvessel in a concentration between about 1.0 or 1.05 and about 4equivalents relative to substrate. In preferred embodiments, thethioester is present in an amount between 1.1 and 2.0 equivalentsrelative to substrate. More preferably, the thioester is present in anamount between 1.1 and 1.7 equivalents.

It has been found that in certain acylations of a substrate using LovDacyltransferase, pH may affect reaction progression. Accordingly, in oneor more embodiments, the acyltransferase reaction occurs at a pH ofgreater than 7. Preferably, the pH is greater than 8. More preferably,the pH is between about 8.0 and 10.5 and most preferably, the pH isbetween about 8.0 and 9.5.

The acylation reaction may be effected at various temperatures,including between 15° C. and 45° C., also between 20° C. and 40° C., andbetween 20° C. and 35° C. Preferably, the reaction occurs at atemperature between 22° C. and 28° C. Most preferably, the reactionoccurs at about 25° C. Preferably, the acylation reaction occurs withstirring. In some instances, a cosolvent may be added to aid in completesolvation of one or more components of the reaction medium or to preventthe formation of intractable slurries. In preferred embodiments, theacylation of monacolin J hydroxy acid, sodium salt or ammonium salt,does not require addition of cosolvent.

In some embodiments, a controlled feed of substrate, such as but notlimited to substrate in solution, is added. For example, a syringe pumpcan be used to deliver a controlled feed of substrate dissolved in asolvent system. In an alternative embodiment, substrate may be added inbatches, for example, an initial charge of substrate is followed at someperiod of time later by a second charge. In yet another embodiment, asingle charge may be followed by a controlled feed of substrate.

Reaction time is preferably of sufficient length to allow completeconversion of substrate to target product. In a preferred embodimentusing monacolin J hydroxy acid, sodium salt in a preferred amount andusing preferred conditions, the reaction is allowed to proceed between 2and 48 hours before workup. In some preferred embodiments, the course ofthe reaction is monitored by suitable methods. The reaction is quenchedor otherwise “worked-up” when monitoring indicates no appreciable degreeof additional conversion of substrate to target product. Isolation mayinvolve extraction. In one preferred embodiment, one or more volumes ofmethyl tert-butyl ether (MTBE) are used to perform a first extractionbefore adjustment of the aqueous phase pH and subsequent extraction withethyl acetate (EtOAc) as described below in the examples. A skilledartisan will appreciate that reaction time and workup conditions forvarious substrates may be determined using known methods and no morethan routine experimentation.

It is contemplated that excess thioester may be recovered from one ormore waste streams. For example, in one embodiment, excess or unreactedthioester is extracted into MTBE. The MTBE extract can then undergo oneor more aqueous washes, preferably basic aqueous washes, to remove thiolbyproduct. The purified thioester may then resubjected to reactionconditions as either a crude recycled material or as a blend with freshthioester. In some embodiments, the recycled thioester is distilled toincrease purity prior to addition to the reaction medium.

The order of addition of materials may vary in one or more embodiments.For example, the following orders of addition may be used: enzyme,scavenger, thioester; scavenger, enzyme, thioester; or scavenger,thioester, enzyme. In embodiments where a scavenger is not utilized,enzyme or thioester may be added first to the reaction medium.

In some embodiments, the process comprises the acylation of asimvastatin precursor using (i) a precipitating agent and/or (ii) asequestering agent. In an aspect of one or more embodiments, a skilledartisan can assess the solubility of simvastatin salts. For example, askilled artisan could generate the sodium, potassium, calcium,magnesium, and ammonium salts of simvastatin or a precursor thereof. Theskilled artisan could then assess these salts in various solventsystems. The skilled artisan would then compare the solubility of thesesalts in a given solvent system against the solubility of thecorresponding monacolin J salt. Monacolin J salts which are soluble inthe given solvent system which produce simvastatin salts which havereduced solubility (or low or no solubility) in the solvent system canthen be used in a process for producing simvastatin which utilizes theprecipitation of product to favorably shift the equilibrium of thereaction.

The various methods and processes described herein are not limited tothe use of any specific LovD acyltransferase enzyme, and can be carriedout with any LovD acyltransferase capable of catalyzing the desired acyltransfer. LovD acyltransferases useful in the processes and methodsdescribed herein include, but are not limited to, the wild-type LovDacyltransferase obtainable from A. terreus (the amino acid sequence ofwhich is provided as SEQ ID NO:2; see also WO 00/037692 and WO2007/139871), the various mutants of this wild-type LovD acyltransferasedescribed in, for example, Biotechnol Bioeng, 2009 Jan. 1; 102(1):20-8,and the various variants described in U.S. Patent Application No.61/247,253, filed Sep. 30, 2009, titled “Variant LovD Polypeptides andTheir Uses” and U.S. Patent Application No. 12/890,134, filed Sep. 24,2010, issued as U.S Pat. No. 8,383,382, both of which are collectivelyreferred to as the “Variant Applications.”

As disclosed in the Variant Applications, numerous variant LovDacyltransferases have been discovered that have improved properties ascompared to the wild-type LovD acyltransferase of SEQ ID NO:2. All ofthese LovD variants can be advantageously used in the methods andprocesses described herein. For example, as disclosed in the aboveapplications, such LovD variant acyltransferases include one or moremutations at selected positions that correlate with one or more improvedproperties, such as increased catalytic activity, increased thermalstability, reduced aggregation and/or increased stability to cell lysisconditions. The variant LovD acyltransferases can include one or moremutations from a single category (for example, one or more mutationsthat increase catalytic activity), or mutations from two or moredifferent categories. By selecting mutations correlating with specificproperties, variant LovD polypeptides suitable for use under specifiedconditions can be readily obtained.

Positions in the wild-type LovD acyltransferase of sequence of SEQ IDNO:2 at which mutations have been found that correlate with one or moreimproved properties, such as increased catalytic activity include, butare not limited to, A123, M157, S164, S172, L174, A178, N191, L192,A247, R250, S256, A261, G275, Q297, L361, V370 and N391. Specific,exemplary mutations of the wild-type LovD acyltransferase of SEQ ID NO:2that correlate with increased catalytic activity include, but are notlimited to, A123P, M157V, S164G, S172N, L174F, A178L, N191G, L192I,A247S, R250K, S256T, A261H, G275S, Q297G, L361M, V370I and N391S.

Additional positions at which mutations have been found which correlatewith one or more improved properties, such as thermal stability,include, but are not limited to, Q241, A261, Q295 and Q412. Specific,exemplary mutations of the wild-type LovD acyltransferase of SEQ ID NO:2that correlate with increased thermal stability include, but are notlimited to, Q241M, A261H, Q295R and Q412R.

Yet further positions at which mutations have been found that correlatewith one or more improved properties, such as reduced aggregation,include but are not limited to, N43, D96 and H404. Specific, exemplarymutations of the wild-type LovD acyltransferase of SEQ ID NO:2 thatcorrelate with reduced aggregation include, but are not limited to,N43R, D96R and H404K.

Still further positions at which mutations were found that correlatedwith one or more improved properties, such as increased stabilityinclude, but are not limited to, C40, C60 and D254. Specific exemplarymutations of the wild-type LovD acyltransferase of SEQ ID NO:2 thatcorrelate with increased stability include, but are not limited to,C40R, C60R and D254E.

Positions were also discovered that could be mutated without detrimentaleffect, whether or not such mutations conferred the LovD acyltransferasewith improved properties. Positions within the wild-type LovDacyltransferase sequence of SEQ ID NO:2 which can be mutated withoutdetrimental effect include, but are not limited to, I4, A9, K26, R28,I35, C40, S41, N43, C60, S109, S142, A184V, N191S, A261, L292, Q297,L335, A377, A383, N391 and H404. Specific, exemplary mutations that canbe incorporated at these positions include, but are not limited to, 14N,A9V, K26E, R28K, R28S, I35L, C40A, C40V, C40F, S41R, N43Y, C60F, C60Y,C60N, C60H, S109C, S142N, A184T, A184V, N191S, A261T, A261E, A261V,L292R, Q297E, L335M, A377V, A383V, N391D and H404R.

LovD acyltransferases having sequences that correspond to SEQ ID NO:2and that include one or more mutations at any of the positions mentionedabove are useful in the methods and processes described herein. In aspecific embodiment, useful LovD acyltransferases have sequences thatcorrespond to SEQ ID NO:2 and include mutations at positions L174 andA178 (in a specific embodiment L174F and A178L), and optionally from 1to about 30 additional mutations, which may be selected form thepositions and residues discussed above. In another specific embodiment,useful LovD acyltransferases have sequences that correspond to SEQ IDNO:2 and include at least the following mutations: A123P, L174F, A178L,N191(S or G), A247S and L361M, and from zero up to about 26 additionalmutations, which may be selected from the various different positionsand mutations discussed above.

As disclosed in the Variant Applications, it is believed that the aminoacid at position 76 may be involved in catalysis. Mutations at thisresidue position should be preferably avoided. It is also believed thatthe amino acids at position 79, 148, 188 and/or 363 may contribute tocatalysis. Mutations at these positions are likewise preferably avoided.

In addition to the mutations described above, useful LovDacyltransferases may also include conservative mutations at one or morepositions (independently of or in addition to the mutations discussedabove). Generally, wild-type and variant LovD acyltransferases includingconservative mutations will contain from 1 to 20 such mutations. Inadditional embodiments, wild-type and variant LovD acyltransferases thatare truncated at one or both termini and that retain their catalyticactivity may be used in the methods and processes described herein. Insome embodiments, such truncated LovD acyltransferases include wild-typeor variant LovD acyltransferases in which 1 to 15 amino acids areomitted from the N-terminus and/or from 1 to 6 amino acids are omittedfrom the C-terminus.

Specific embodiments of variant LovD acyltransferases having improvedcatalytic activity as compared to the wild-type acyltransferase of SEQID NO:2 that are useful in the methods and processes described hereinare provided in Table 2, below:

TABLE 2 Variant No. Mutations (Relative to SEQ ID NO: 2) Activity* 120A123P; L174F; A178L; N191S; A247S; L361M; + 4 I35L; A123P; L174F; A178L;N191S; A247S; L361M; + 6 A123P; L174F; A178L; N191S; A247S; G275S;L361M; + 8 A123P; L174F; A178L; N191S; A247S; R250K; L361M; + 10 A123P;L174F; A178L; N191S; A247S; Q297E; L361M; + 12 R28K; A123P; L174F;A178L; N191S; A247S; L361M; + 14 A123P; L174F; A178L; A184T; N191S;A247S; L361M; + 16 A123P; L174F; A178L; N191S; A247S; Q297E; L361M; + 18A123P; L174F; A178L; N191S; L192I; A247S; L361M; + 20 A123P; L174F;A178L; N191S; A247S; R250K; L361M; + 22 A123P; L174F; A178L; N191S;A247S; A261E; L361M; + 24 A123P; L174F; A178L; N191S; A247S; L361M;H404R; + 26 K26E; A123P; L174F; A178L; N191S; A247S; L361M; + 28 A123P;S172N; L174F; A178L; N191S; A247S; G275S; L361M; ++ 30 A123P; M157V;S172N; L174F; A178L; N191S; A247S; G275S; L361M; ++ 32 A123P; L174F;A178L; N191G; A247S; G275S; L361M; + 34 A123P; L174F; A178L; N191S;A247S; G275S; L335M; L361M; + 36 A123P; L174F; A178L; N191S; A247S;G275S; L361M; H404K; + 38 A123P; L174F; A178L; A184V; N191S; A247S;G275S; L361M; + 40 D96R; A123P; L174F; A178L; N191S; A247S; G275S;L361M; + 42 A123P; L174F; A178L; N191G; A247S; G275S; L361M; + 44 A123P;L174F; A178L; N191S; A247S; G275S; L335M; L361M; + 46 A123P; L174F;A178L; N191S; A247S; G275S; L292R; L361M; + 48 A123P; L174F; A178L;N191S; L192I; A247S; R250K; G275S; Q297E; L361M; ++ 50 A123P; L174F;A178L; N191S; L192I; A247S; R250K; G275S; L361M; ++ 52 K26E; C40R; N43Y;A123P; L174F; A178L; N191S; L192I; A247S; G275S; ++ L361M; 54 K26E;C40R; A123P; L174F; A178L; N191S; L192I; A247S; G275S; L361M; ++ 56K26E; A123P; L174F; A178L; N191S; A247S; G275S; L361M; + 58 A9V; K26E;A123P; M157V; S172N; L174F; A178L; N191S; L192I; A247S; +++ R250K;G275S; Q297E; L361M; A383V; 60 K26E; A123P; M157V; S172N; L174F; A178L;N191S; L192I; A247S; R250K; +++ G275S; L361M; 62 A123P; M157V; S172N;L174F; A178L; N191G; A247S; G275S; L335M; ++ L361M; 64 N43R; D96R;A123P; M157V; S172N; L174F; A178L; N191S; A247S; G275S; ++ L361M; H404K;66 A9V; K26E; A123P; M157V; S172N; L174F; A178L; N191S; L192I; A247S;+++ R250K; S256T; G275S; Q297E; L361M; A383V; 68 A9V; K26E; S41R; A123P;M157V; S172N; L174F; A178L; N191S; L192I; +++ A247S; R250K; A261V;G275S; Q297E; L361M; A383V; 70 A9V; K26E; R28K; A123P; M157V; S164G;S172N; L174F; A178L; N191G; +++ L192I; Q241M; A247S; R250K; G275S;Q297E; L361M; V370I; A383V; 72 A9V; K26E; R28K; C40R; A123P; M157V;S164G; S172N; L174F; A178L; +++ N191G; L192I; Q241M; A247S; R250K;G275S; Q297E; L361M; V370I; A383V; 74 A9V; K26E; R28K; C40R; A123P;M157V; S164G; S172N; L174F; A178L; +++ N191G; L192I; Q241M; A247S;R250K; G275S; Q297E; L361M; V370I; A383V; 76 A9V; K26E; A123P; M157V;S164G; S172N; L174F; A178L; N191G; L192I; +++ A247S; R250K; G275S;Q297E; L361M; V370I; A377V; A383V; 78 A9V; K26E; N43R; A123P; M157V;S164G; S172N; L174F; A178L; N191G; ++++ L192I; Q241M; A247S; R250K;G275S; Q297E; L361M; V370I; A383V; H404K; 80 A9V; K26E; N43R; D96R;A123P; M157V; S164G; S172N; L174F; A178L; ++++ N191G; L192I; Q241M;A247S; R250K; G275S; Q297E; L361M; V370I; A383V; H404K; 82 A9V; K26E;D96R; A123P; M157V; S164G; S172N; L174F; A178L; N191G; ++++ L192I;Q241M; A247S; R250K; G275S; Q297E; L361M; V370I; A383V; 84 A9V; K26E;D96R; A123P; M157V; S172N; L174F; A178L; N191G; L192I; +++ Q241M; A247S;R250K; G275S; Q297E; L361M; V370I; A383V; 86 A9V; K26E; N43R; D96R;A123P; M157V; S164G; S172N; L174F; A178L; +++ N191G; L192I; A247S;R250K; G275S; Q297E; L361M; V370I; A383V; H404K; 88 A9V; K26E; N43R;D96R; A123P; M157V; S164G; S172N; L174F; A178L; ++++ N191G; L192I;Q241M; A247S; R250K; G275S; Q297E; L361M; V370I; A383V; 90 A9V; K26E;R28S; N43R; A123P; M157V; S164G; S172N; L174F; A178L; ++++ N191G; L192I;Q241M; A247S; R250K; D254E; G275S; Q297E; L361M; V370I; A383V; H404K; 92A9V; K26E; N43R; A123P; M157V; S164G; S172N; L174F; A178L; N191G; ++++L192I; Q241M; A247S; R250K; A261V; G275S; Q295R; Q297E; L361M; V370I;A383V; H404K; Q412R; 94 A9V; K26E; N43R; A123P; M157V; S164G; S172N;L174F; A178L; N191G; ++++ L192I; Q241M; A247S; R250K; A261V; G275S;Q297E; L361M; V370I; A383V; H404K; 96 A9V; K26E; N43R; A123P; M157V;S164G; S172N; L174F; A178L; N191G; ++++ L192I; Q241M; A247S; R250K;A261V; G275S; Q295R; Q297E; L361M; V370I; A383V; N391D; H404K; 98 A9V;K26E; N43R; A123P; M157V; S164G; S172N; L174F; A178L; N191G; ++++ L192I;Q241M; A247S; R250K; S256T; A261V; G275S; Q297G; L361M; V370I; A383V;N391S; H404K; 100 A9V; K26E; N43R; A123P; M157V; S164G; S172N; L174F;A178L; N191G; ++++ L192I; Q241M; A247S; R250K; S256T; A261V; G275S;Q297G; L361M; V370I; A383V; N391S; H404K; 102 A9V; K26E; N43R; S109C;A123P; M157V; S164G; S172N; L174F; A178L; ++++ N191G; L192I; Q241M;A247S; R250K; S256T; A261V; G275S; Q297G; L361M; V370I; A383V; N391S;H404K; 104 A9V; K26E; N43R; A123P; M157V; S164G; S172N; L174F; A178L;N191G; ++++ L192I; Q241M; A247S; R250K; S256T; A261H; G275S; Q297G;L361M; V370I; A383V; N391S; H404K; 106 A9V; K26E; N43R; A123P; M157V;S164G; S172N; L174F; A178L; N191G; ++++ L192I; Q241M; A247S; R250K;S256T; A261H; G275S; Q295R; Q297G; L361M; V370I; A383V; N391S; H404K;Q412R; 108 I4N; A9V; K26E; R28S; N43R; A123P; M157V; S164G; S172N;L174F; A178L; ++++ N191G; L192I; Q241M; A247S; R250K; S256T; A261H;G275S; Q297G; L361M; V370I; A383V; N391S; H404K; 110 I4N; A9V; K26E;R28S; N43R; A123P; M157V; S164G; S172N; L174F; A178L; ++++ N191G; L192I;Q241M; A247S; R250K; D254E; S256T; A261H; G275S; Q297G; L361M; V370I;A383V; N391S; H404K; 112 I4N; A9V; K26E; R28S; N43R; S109C; A123P;M157V; S164G; S172N; L174F; ++++ A178L; N191G; L192I; Q241M; A247S;R250K; S256T; A261H; G275S; Q295R; Q297G; L361M; V370I; A383V; N391S;H404K; Q412R; 114 I4N; A9V; K26E; R28S; I35L; N43R; D96R; A123P; M157V;S164G; S172N; ++++ L174F; A178L; N191G; L192I; Q241M; A247S; R250K;S256T; A261H; G275S; Q297G; L335M; L361M; V370I; A383V; N391S; H404K;116 I4N; A9V; K26E; R28S; I35L; N43R; D96R; S109C; A123P; M157V; S164G;++++ S172N; L174F; A178L; N191G; L192I; Q241M; A247S; R250K; S256T;A261H; G275S; Q297G; L335M; L361M; V370I; A383V; N391S; H404K; 118 I4N;A9V; K26E; R28S; I35L; C40R; N43R; C60R; D96R; S109C; A123P; ++++ M157V;S164G; S172N; L174F; A178L; N191G; L192I; Q241M; A247S; R250K; D254E;S256T; A261H; G275S; Q297G; L335M; L361M; V370I; A383V; N391S; H404K;*Activity relative to the wild-type LovD acyltransferase of SEQ ID NO:2. A relative activity of “+” exhibited from about 10 to 50-fold greateractivity than wild-type; variants with a relative activity of “++”exhibited from about 50 to 100-fold greater activity than wild type;variants with a relative activity of “+++” exhibited from about 100 to500-fold greater activity than wild type; and variants with a relativeactivity of “++++” exhibited from about 500 to 2000-fold greateractivity than wild type

7. EXAMPLES

The following Examples are illustrative of one or more embodiments andas such are not to be considered as limiting the scope of the claimsappended hereto.

Example 1 Preparation of Methyl 3-(2,2-Dimethylbutanoylthio)propionate

A solution of N,N-diisopropylethylamine (19.9 mL, 120 mmol) and methyl3-mercaptopropanoate (7.21 60 mmol) in isopropyl acetate (i-PrOAc, 100mL) was cooled to an internal temperature of 2° C. (brine ice bath). Tothis vigorously stirred solution was added 2,2-dimethylbutanoyl chloride(8.1 g, 60 mmol) dropwise over 10 min. The resulting suspension was thenstirred at 25° C. for 2 h. The reaction was monitored by checking thedisappearance of methyl 3-mercaptopropanoate using thin-layerchromatography (TLC) on silica plates. Spots were stained with iodine(eluent: 5% EtOAc/heptane; R_(f) of methyl 3-mercaptopropanoate: 0.20).The reaction was then quenched by addition of saturated ammoniumchloride (100 mL) followed by i-PrOAc (100 mL) and the resultant mixturewas stirred until all solid dissolved. The phases were separated and theorganic phase was washed successively with 1% aqueous hydrochloric acid(100 mL) and then water (2×50 mL). The organic phase was then dried oversodium sulfate, filtered, and concentrated under reduced pressure (45°C. bath, 50 mm Hg) to obtain a crude mixture as a pale yellow liquid.The crude mixture was subjected to column chromatography over silica gelusing a heptane to 2% EtOAc:heptane gradient. Fractions comprising thepure product were combined and concentrated to afford 10.5 g (80%) ofmethyl 3-(2,2-dimethylbutanoylthio)propionate.

Example 2 Preparation of Monacolin J Hydroxy Acid from Lovastatin

To lovastatin (30 g, 0.074 mol) in a 3-neck round bottom flask (RBF)fitted with a condenser was added isopropanol (IPA, 250 mL). KOH pellets(33.2 g, 0.593 mol) and water (3 mL, 0.1 vol) were then added to thestirred suspension. The reaction was stirred at 80° C. (internaltemperature) for 7 h. The reaction was then cooled to ˜50° C. and IPAwas removed under reduced pressure (35° C., 50 mbar) until a finalvolume of ˜100 mL (3.3 vol) was achieved. Water (110 mL, 3.7 vol) wasadded to the residue and the solution was cooled to ˜10° C. in anice-water bath. 6 M HCl (92 mL, 3.0 vol) was added dropwise to thesolution while maintaining the internal temperature between 12-17° C.The pH of the solution was thereby adjusted to a final pH between 3 and4. The mixture was then stirred in an ice-bath for 2 h. The solidobtained was filtered off and washed with water (60-90 mL, 2-3 vol) andthen with heptane (60 mL, 2 vol). The filter cake was dried in a vacuumoven at 25° C. for 24 h to yield a white solid (22.4 g, 90% yield)with >99% purity by HPLC analysis.

Example 3 Enzymatic Preparation of Simvastatin Hydroxy Acid, Sodium Saltfrom Monacolin J Hydroxy Acid, in the Absence of a Thiol Scavenger

The reaction was run in a 250 mL 3-neck RBF using an overhead stirrerfitted with a flat-blade impeller and an internal thermometer. Executionof the following procedure provided 70-80% of simvastatin hydroxy acid,sodium salt in a single crop as a white solid with a chemical purity ofat least 96% according to HPLC analysis. To the RBF was charged,sequentially: monacolin J hydroxy acid (10 g), 1M NaOH (32.5 mL) anddeionized water (13 mL). The mixture was stirred until all solid wasdissolved prior to the addition of buffer (triethanolamine, 400 mM,pH=8.5, 66 mL). The pH of the resultant mixture was then adjusted from9.5 to 8.5 with 5 M HCl (1.2 mL) prior to the addition of enzyme. A pHof at least 7.5 was maintained during the course of the reaction. Enzyme(˜0.6-0.75 g of LovD acyltransferase or an equivalent amount of avariant thereof) was charged to the stirred mixture as a powder. Themixture was stirred until homogenous.

Thioester (methyl 3-(2,2-dimethylbutanoylthio)-propanoate, 11 mL) wasadded and the resulting biphasic mixture was stirred at 240 rpm at 25°C. (internal temperature). The reaction course was followed periodicallyby taking samples from the reaction mixture, quenching, and analyzing.Additional enzyme (0.127 g of LovD or an equivalent amount of a variantthereof) was charged to the stirred mixture after 7 h. After analysisindicated maximum conversion (60-70 hours), the pH of the reactionmixture was adjusted to 9.0 from 7.8 using 10 M NaOH solution (1.6 mL)and the reaction mixture was agitated at 345 rpm for 10 minutes.

Workup of the reaction mixture may be accomplished by first extractingthe aqueous phase with methyl tert-butyl ether (MTBE). After extractionwith MTBE (2 extractions), the aqueous phase was adjusted to a pH ofabout 5.3 to 5.4 using 0.5 M HCl while maintaining a temperature below20° C. The aqueous phase is extracted three times with EtOAc. Duringeach extraction, additional HCl was added, as needed, to maintain the pHof the aqueous phase between 5.3 and 5.4. The EtOAc phases from the 3extractions were combined. The combined ethyl acetate extracts werefiltered through a pad of Celite® (1 g) in a standard G4 sintered glassfunnel under reduced pressure to clarify the extract. The filter cakewas washed with ethyl acetate (10 mL) and the washings were combinedwith the filtrate. The filtrate was concentrated as needed or additionalEtOAc was added to produce an EtOAc solution with a volume of about 160mL. This EtOAc solution was used for the next step.

The reaction can be monitored by any suitable method. One suitable andexemplary method included monitoring by HPLC analysis. For example, a 5μL, aliquot of the reaction mixture was taken and dissolved in 1.0 mL ofMeCN:water (95:5). The sample was centrifuged to remove precipitatedenzyme and the supernatant was analyzed on a Zorbax Eclipse® C18 column(150×4.6 mm, 5 μm) using a mobile phase gradient of water and 0.1% TFAto MeCN and 0.1% TFA. A sample flow rate of 2.0 mL/min with a detectionwavelength of 238 nm, a column temperature of 30° C., and an injectionvolume of 10 μL was used. Percent conversion was calculated by dividingthe sum of the area of detectable simvastatin hydroxy acid andsimvastatin over the sum of the areas of detectable monacolin J hydroxyacid, monacolin J lactone, simvastatin hydroxy acid and simvastatin.Monacolin J hydroxy acid and simvastatin hydroxy acid both demonstrateda response factor of 1.

Example 4 Preparation of Simvastatin Hydroxy Acid, Ammonium Salt fromSimvastatin Hydroxy Acid, Sodium Salt

160 mL of ethyl acetate solution containing simvastatin hydroxy acid,sodium salt—from Example 3—was charged to a 250 mL 3-neck RBF and thereaction mixture was stirred at 250 rpm at 21° C. A 1:1 (v/v) mixture ofammonium hydroxide (5 mL) and MeOH (5 mL) was then added dropwise over10 mins to the reaction mixture while maintaining the internaltemperature at 21-22° C. After complete addition of the ammoniumhydroxide and MeOH mixture, the stirrer speed was increased and themixture was agitated for 1 h at 21° C. and then for 1 h at 0-5° C. Thewhite solid was then filtered through a standard G4 sintered glassfunnel under vacuum and the reaction vessel and filter cake were rinsedwith cold EtOAc. The white solid was dried in a vacuum oven (2 mm Hg) at25° C. for 24 h. This provided 10.48 g (78.2%) isolated yield ofsimvastatin hydroxy acid, ammonium salt as a white solid with a chemicalpurity of >96% by HPLC analysis.

Example 5 Enzymatic Preparation of Simvastatin Hydroxy Acid, Sodium Saltfrom Monacolin J Hydroxy Acid, in the Presence of a Thiol Scavenger

A 250 mL 3-neck RBF was equipped with an overhead stirrer, a flat-bladeimpeller and an internal thermometer. The reaction vessel was chargedsequentially with the following: monacolin J hydroxy acid (5 g), 1M NaOH(16.3 mL), and deionized water (6.7 mL). The mixture was stirred untilall solid was dissolved prior to the addition of buffer(triethanolamine, 400 mM, pH=8.5, 33.3 mL). The pH of the resultantmixture was adjusted from 9.6 to 9.0 with 5 M HCl (0.12 mL) prior to theaddition of enzyme. Enzyme (0.10 g of acyltransferase LovD or anequivalent amount of a variant thereof) was charged to the stirredmixture as a powder. The mixture was stirred until homogeneous.Activated charcoal (0.67 g) was subsequently added and the mixture wasstirred for another 5 minutes at 240 rpm at 25° C.

Thioester (methyl 3-(2,2-dimethylbutanoylthio)-propanoate, 5.5 mL) wasthen added to start the enzymatic reaction. The resulting mixture wasstirred at 240 rpm at 25° C. (internal temperature). The reaction coursewas followed periodically by taking samples from the reaction mixture,quenching, and analyzing using HPLC. After analysis indicated maximumconversion (24 h), the reaction mixture was filtered through a pad ofCelite (1.5 g) in a standard G4 sintered glass funnel under reducedpressure to remove the charcoal. The 250 mL 3-neck RBF was rinsed withdeionized water (5 mL), which was filtered through the same pad ofCelite® and then combined with the filtrate. The filter cake was washedwith water (5 mL) and the washings were collected and combined with thefiltrate. The filtrate was recharged into a new 250 mL 3-neck flask andthe pH of the filtrate was adjusted to 9.0 from 8.2 using 10 M NaOHsolution (0.55 mL).

Workup of the reaction mixture was accomplished by first extracting theaqueous phase with methyl tert-butyl ether. After extraction with MTBE(2 extractions), the aqueous phase was adjusted to a pH of about 5.3 to5.4 using 0.5 M HCl while maintaining a temperature below 20° C. Theaqueous phase was extracted three times with EtOAc. During eachextraction, additional HCl was added, as needed, to maintain the pH ofthe aqueous phase between 5.3 and 5.4. The EtOAc phases from the 3extractions were combined. The combined ethyl acetate extracts werefiltered through a pad of Celite® (1 g) in a standard G4 sintered glassfunnel under reduced pressure to clarify the extract. The filter cakewas washed with ethyl acetate (10 mL) and the washings were combinedwith the filtrate. The filtrate was concentrated as needed or additionalEtOAc was added to produce an EtOAc solution with a volume of about 65mL. This concentrated EtOAc solution was used for the next step, theformation of simvastatin hydroxy acid, ammonium salt, according to themethod of example 4.

Example 6 General Method for the Improved Enzymatic Acylation of aSubstrate Using a Thioester and a LovD Acyltransferase

To a suitable reaction medium was charged a substrate having a freehydroxyl substituent. Solvent and buffer (as needed) were added. Next,while stirring, thioester (1.1-2.0 eq.) and activated charcoal (˜10 g/Lof solvent) were added. After homogenization by stirring, theacyltransferase enzyme LovD or a variant thereof (0.01 to 0.2 eq.) wereadded. The reaction was stirred until completion. Workup includedfiltration, extraction, and/or concentration to produce the finalacylated product.

Example 7 Conversion of Monacolin J Hydroxy Acid to Simvastatin HydroxyAcid Ammonium Salt and Isolation of Simvastatin Hydroxy Acid AmmoniumSalt

A 250 mL 3-neck round bottom flask (RBF) was equipped with an overheadstirrer, a flat-blade impeller and an internal thermometer. The reactionvessel was charged with monacolin J hydroxy acid (10 g, 29.58 mmol).Deionized water (112.0 mL) and NH₄OH (4.2 mL) were added subsequently.The mixture was stirred until all solid dissolved prior to the pHadjustment (˜2 min). The pH of the resultant mixture was adjusted from9.2 to 9.0 with 5 M HCl (1.5 mL) prior to the addition of enzyme. LovDenzyme (0.10 g) was charged to the stirred mixture as a powder. Themixture was stirred for 5 minutes at 300 rpm at 25° C. to obtainhomogeneity. DMB-S-MMP (methyl 3-(2,2-dimethylbutanoylthio)-propanoate,7.1 mL, 32.54 mmol, 1.1 eq) was added to start the enzymatic reaction.The resulting biphasic mixture was stirred at 300 rpm at 25° C.(internal temperature). The pH of the reaction was controlled at 9.0 bypH stat and titration with 25% NH₄OH solution. Approximately 97%conversion was obtained after 48 h.

Simvastatin hydroxy acid ammonium salt could be isolated from the abovereaction as follows. After in-process analysis indicated maximumconversion, the reaction mixture was filtered through a standard G4sintered glass funnel under reduced pressure. The 250 mL 3-neck RBF wasrinsed with chilled deionized water (10 mL) and the slurry was filteredthrough the same sintered glass funnel. The filter cake was washed twicewith chilled deionized water (20 mL) and then washed three times withMTBE (40 mL). The white solid was dried in a vacuum oven (2 mmHg) at 25°C. for 24 h to afford approximately 11.4 to 11.7 g (85 to 87% isolatedyield) of simvastatin hydroxy acid ammonium salt as a white solid with achemical purity of about 97 to 98% (AUC, 238 nm). In one embodiment, thevariant having the mutations described in SEQ ID NO:116 provides goodresults according to the above conditions. Reaction conditions for theabove examples, including loading amounts of substrate or enzyme, mayneed to be optimized for the reactivity profile of other variants.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. A method of making a statin compound, comprisingcontacting a LovD acyltransferase substrate with a LovD acyltransferase,wherein said LovD acvltransferase is a recombinant variant comprisingSEQ ID NO: 2 that includes the mutations L174F and A178L and additional1 to 30 additional mutations in the presence of a thioester donor and anagent selected from a thiol scavenger, a precipitating agent andcombinations thereof, under conditions which yield a statin compound. 2.The method of claim 1 in which the LovD acyltransferase substrate ismonacolin J.
 3. The method of claim 2, in which the monacolin J is amonacolin J hydroxy acid salt.
 4. The method of claim 2 in which themonacolin J is monacolin J lactone.
 5. The method of claim 1 in whichthe LovD acyltransferase substrate is lovastatin.
 6. The method of claim5 in which the lovastatin is a lovastatin hydroxy acid salt.
 7. Themethod of claim 5 in which the lovastatin is lovastatin lactone.
 8. Themethod of claim 1, in which the LovD acyltransferase substrate is6-hydroxy-6-des-methyl-monacolin-J.
 9. The method of claim 8, in whichthe 6-hydroxyl-6-desmethyl-monacolin J is a 6-hydroxy-6-des-methylmonacolin J hydroxy acid salt.
 10. The method of claim 8, in which the6-hydroxy-6-desmethyl monacolin J is 6-hydroxyl-6-des-methyl monacolin Jlactone.
 11. The method of claim 1 in which the LovD acyltransferasesubstrate is pravastatin.
 12. The method of claim 11 in which thepravastatin is a pravastatin hydroxy acid salt.
 13. The method of claim11 in which the pravastatin is a pravastatin lactone.
 14. The method ofclaim 1, in which the thioester donor is an alpha-dimethylbutyrylthioester.
 15. The method of claim 14 in which the alpha-dimethylbutyrylthioester is selected from the group consisting ofα-dimethylbutyryl-S-methyl-mercaptopropionate (DMB-S-MMP),dimethylbutyryl-S-ethyl mercaptopropionate (DMB-S-EMP),dimethylbutyryl-S-methyl thioglycolate (DMB-S-MTG),dimethylbutyryl-S-methyl mercaptobutyrate (DMB-S-MMB),S-2-acetamidoethyl 2,2-dimethylbutanethioate, S-acetamidomethyl2,2-dimethylbutanethioate and methyl2-(2,2-dimethylbutanoylthio)acetate.
 16. The method of claim 15 in whichthe alpha-dimethylbutyryl thioester is DMB-S-MMP.
 17. The method ofclaim 14, which is carried out in an aqueous medium at a pH in the rangeof about pH 8 to pH 9.5.
 18. The method of claim 14, which is carriedout in an aqueous buffer having an initial pH of about pH
 9. 19. Themethod of claim 14, in which the agent is a thiol scavenging agent. 20.The method of claim 19, in which the thiol scavenging agent is activatedcharcoal.
 21. The method of claim 14, in which the agent is aprecipitating agent.
 22. The method of claim 21, in which theprecipitating agent is ammonium hydroxide.
 23. The method of claim 14,in which the LovD acyltransferase is one of the LovD variant selectedfrom the group consist of the variants shown Table
 2. 24. The method ofclaim 14, in which the LovD acyltransferase is a LovD variant comprisingSEQ ID NO: 2 that includes the mutations L174F and A178L.
 25. A methodof making simvastatin hydroxy acid ammonium salt comprising contactingmonacolin J or lovastatin with a LovD acyltransferase, wherein said LovDacvltransferase is a recombinant variant comprising SEQ ID NO: 2 thatincludes the mutations L174F and A178L and additional 1 to 30 additionalmutations in the presence of an alpha-dimethylbutyryl thioester donorand ammonium hydroxide under conditions which yield simvastatin hydroxyacid ammonium salt.
 26. The method of claim 25, which is carried out inaqueous solution at a pH in the range of about pH 8 to pH 9.5.
 27. Themethod of claim 25, which is carried out in an aqueous buffer having aninitial pH of about pH
 9. 28. The method of claim 25 in which the saidLovD variant comprises SEQ ID NO: 2 that includes L174F and A178L. 29.The method of claim 28, further comprising the following mutationsA123P; N191S/G; A247S and L361M.